The present invention is related to computer systems for analyzing polymers. More particularly, the invention provides systems and methods for analyzing biopolymers, such as nucleic acids, in order to identify monomer substitutions, insertions and deletions.
U.S. Pat. No. 5,424,186, which is hereby incorporated by reference for all purposes, describes pioneering techniques for, among other things, forming and using high density arrays of molecules such as oligonucleotides, peptides, polysaccharides, and other materials. Arrays of oligonucleotides, for example, are formed on the surface by sequentially removing a photoremovable group from a surface, coupling a monomer to the exposed region of the surface, and repeating the process. These techniques have been used to form extremely dense arrays of oligonucleotides, peptides, and other materials. Such arrays are useful in, for example, drug development, oligonucleotide sequencing, oligonucleotide sequence checking, and a variety of other applications. The synthesis technology associated with this invention has come to be known as xe2x80x9cVLSIPSxe2x80x9d or xe2x80x9cVery Large Scale Immobilized Polymer Synthesisxe2x80x9d technology.
Additional techniques for forming and using such arrays are described in U.S. Pat. No. 5,384,261, which is also incorporated by reference for all purposes. Such techniques include systems for mechanically protecting portions of a substrate (or chip), and selectively deprotecting/coupling materials to the substrate. These techniques are now known as xe2x80x9cVLSIPS II.xe2x80x9d Still further techniques for array synthesis are provided in U.S. application Ser. No. 08/327,512, also incorporated herein by reference for all purposes.
Dense arrays fabricated according to these techniques are used, for example, to screen the array of probes to determine which probe(s) are complementary to a target of interest. According to one specific aspect of the inventions described above, the array is exposed to a labeled target. The target may be labeled with a wide variety of materials, but an exemplary label is a fluorescein label. The array is then scanned with a confocal microscope based detection system, or other related system, to identify where the target has bound to the array. Other labels include, but are not limited to, radioactive labels, large molecule labels, and others.
Innovative computer-aided techniques for identifying monomers in sample polymers are disclosed in U.S. patent application Ser. No. 08/531,137 (attorney docket no. 16528X008210), No. 08/528,656 (attorney docket no. 16528X-017600), and No. 08/618,834 (attorney docket no. 16528X-016400), which are all hereby incorporated by reference for all purposes. However, improved systems and methods are still needed to evaluate, analyze, and process the vast amount of information now used and made available by these pioneering technologies.
One area that can be more thoroughly explored is identifying changes in a heterogeneous sample of polymers. For example, biopsies from cancerous areas or tumors of a patient""s body will typically include genetic material from both normal cells and cancerous cells. In order to better diagnose a cancerous area, it would be beneficial to be able to identify mutations in the p53 genes of a heterogeneous sample, especially where an unknown quantity of wild-type p53 genes are present.
The present invention provides techniques for detecting monomer changes in a heterogeneous sample when an unknown quantity of expected (e.g., wild-type) monomers may also be present. Heterogeneous and homogenous samples are exposed to polymer probes for hybridization, where the homogeneous sample acts as a reference. The hybridization affinities of the polymer probes to the heterogeneous and homogeneous samples are then compared to determine differences between the polymers in the samples. For example, embodiments of the invention allow for the detection of deletion, substitution and insertion mutations in a heterogeneous samples of nucleic acids. Several embodiments of the invention are as follows.
In one embodiment of the invention, a method of analyzing a heterogeneous sample of nucleic acids is provided. Hybridization affinities of a homogeneous sample of nucleic acids to a plurality of nucleic acid probes are received. Hybridization affinities of the heterogeneous sample of nucleic acids to the plurality of nucleic acid probes are also received. The hybridization affinities of the homogeneous and heterogeneous samples are then compared to identify a mutation in the heterogeneous sample. In a preferred embodiment, a first ratio of a hybridization affinity of a non-wild-type probe to a hybridization affinity of a wild-type probe for the homogeneous sample of nucleic acids is calculated and a second ratio of a hybridization affinity of a non-wild-type probe to a hybridization affinity of a wild-type probe for the heterogeneous sample of nucleic acids is calculated. A mutation is identified in the heterogeneous sample if the first ratio is less than the second ratio.
In another embodiment of the invention, a method of analyzing a heterogeneous sample of nucleic acids is provided. Hybridization affinities of a homogeneous sample of nucleic acids to a plurality of nucleic acid probes are received. The plurality of nucleic acid probes include a wild-type probe and at least one non-wild-type probe. Hybridization affinities of a heterogeneous sample of nucleic acids to the plurality of nucleic acid probes are also received. A first ratio of a hybridization affinity of a wild-type probe to a hybridization affinity of a non-wild-type probe for the homogeneous sample of nucleic acids is calculated. A second ratio of a hybridization affinity of a wild-type probe to a hybridization affinity of a non-wild-type probe for the heterogeneous sample of nucleic acids is calculated. A third ratio of the difference between the first and second ratios to the first ratio is then calculated. It is determined that there is a mutation in the heterogeneous sample if the third ratio is above a predetermined threshold, the mutation being identified by the non-wild-type probe.
A further understanding of the nature and advantages of the inventions herein may be realized by reference to the remaining portions of the specification and the attached drawings.